Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase

Lanyon-Hogg, T.; Ritzefeld, M.; Zhang, L.; Pogranyi, B.; Mondal, M.; Johnston, C. D.; Coupland, C. E.; Sefer, L.; Andrei, S. A.; Newington, J.; Magee, A. I.; Siebold, C.; Tate, E. W.

doi:10.1101/2020.10.16.342477

bioRxiv · 10.1101/2020.10.16.342477

Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase

Lanyon-Hogg, T.·Ritzefeld, M.·Zhang, L.·Pogranyi, B.·Mondal, M.·Johnston, C. D.·Coupland, C. E.·Sefer, L.·Andrei, S. A.·Newington, J.·Magee, A. I.·Siebold, C.·Tate, E. W.

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Abstract

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor IMP-1575, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

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Lanyon-Hogg, T., Ritzefeld, M., Zhang, L., Pogranyi, B., Mondal, M., Johnston, C. D., Coupland, C. E., Sefer, L., Andrei, S. A., Newington, J., Magee, A. I., Siebold, C., Tate, E. W.. 2020-10-17. Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase. https://doi.org/10.1101/2020.10.16.342477

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